Analysis of MiRNA-17 and MiRNA-146 Expression During Differentiation of Spermatogonial Stem Like Cells Derived from Mouse Bone Marrow Mesenchymal Stem Cells

In vitro derivation of germ cells from different stem cell sources has been challenging in the treatment of male infertility. MicroRNAs (miRNAs) have an essential role in gene expression at post-transcriptional level. The aim of this research was to find more about miRNA-17 and miRNA-146 expression during differentiation of spermatogonial stem cell like cells (SSC like cells) from mouse bone marrow mesenchymal stem cells (BMSCs) through bone morphogenic protein 4 (BMP4) and retinoic acid (RA) induction. BMSCs were treated with BMP4 to produce primordial germ cell like cells (PGC like cells). The cells were differentiated into SSC like cells by an inducer cocktail including RA, leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF). The PGC like cells and SSC like cells were evaluated for pluripotency (Nanog, Oct-4) and germ cell specific gene (Piwil2, Plzf, Dazl, and Stra8) expression, protein expression (Plzf, Stra8), and miRNA-17 and miRNA-146 mRNA expression. Our results showed that BMP4 leads to Dazl upregulation and Nanog downregulation expression in PGC like cells. RA upregulated Stra8 and Piwil2, and downregulated Nanog and Oct-4. MiRNA-17 and miRNA-146 expression decreased significantly in SSC like cells after RA treatment. This research indicated the aberrant miRNA-17 and miRNA-146 expression in SSC like cells in comparison with SSCs. Downregulation of the two miRNAs using RA in the stimulated undifferentiated state could probably be one of the key factors of SSC like cell arrest.

ver the years, significant number of investigations has been focused on male infertility problems based on stem cell therapy (1). sources of stem cells has been studied to solve male infertility (2). Bone marrow mesenchymal stem cells (BMSCs) are known widely as appropriate stem cells that have capacity to self-renew and differentiate into several cell types such as bone, cartilage, fat, muscle, tendon, liver, and perimordial germ cells (PGCs) (3). Numerous in vitro reports have supported the ability of mesenchymal stem cells (MSCs) to generate germ cells (4,5). The recent findings have reported a variety of inducers and culture systems to reach in vitro germ cells (6,7).

In vitro derivation of germ cells from different
It has been proposed that bone morphogenetic protein 4 (BMP4) and retinoic acid (RA) could promote MSCs transdifferentiation into germ cells (8,9). It is believed that BMP4 signaling has a crucial role in germ cell specification in both in vivo and in vitro approaches (10). Another critical regulator of male germ cell fate RA is important for both the initiation of differentiation, and the entry into meiosis in male germ cells (11). Despite this impressive body of investigations, in vitro-derived or arrest of male germ cell in specific stage which could be due to inefficient cell population, and poorly-defined regulatory mechanisms involved in gene expression control in male germ cell differentiation process.
MicroRNAs (miRNAs) are an important class of non-coding RNAs (ncRNAs) involved in gene expression regulation at post-transcriptional level (12). Mature miRNAS are single-stranded 19-25 nucleotides incorporated into RNA-induced silencing complex, and act through targeting the 3´untranslated region (3´UTR) of mRNAs resulting in translational repression and/or target mRNA degradation (13). The importance of miRNAmediated translational repression in spermatogenesis process has been investigated by deletion of dicer1 as a crucial agent for miRNA biogenesis (14)(15)(16). Notably, impaired biogenesis of miRNAs could disrupt spermatogenesis and lead to infertility in male mice (17). miRNA-17 is a member of Mir-17-92 cluster that modulates pluripotency network (18).
Deregulation of this cluster plays a role in cancer progression (19). More recently, Tong et al. have identified miRNA-17-92 cluster as a regulatory factor in germ cell development (20).

Isolation and culture of mouse BMSCs
Bone marrow aspirated from mouse (NMRI 6-

Immunocytochemistry of BMSCs
Immunocytochemistry was performed to

Induction of osteogenic and adipogenic differentiation
To study the differentiation potential of

Induction of PGC like cell differentiation
For PGC differentiation, confluent BMSCs at passage 3 were cultured at concentration of 1×10 6 cells in 25 cm 2 culture flasks at 37 °C in humidified atmosphere with 5% CO 2 in differentiation medium containing 25 ng/ml BMP4 for 4 days.

Induction of SSC like cell differentiation
After BMP4 treatment, RA (10 -5 M), LIF (1000 U/ml) and bFGF (1 ng/ml) were added for a culture period of 14 days. The medium was changed every 4 days.

Statistical analysis
Statistical analysis was performed using SPSS (v. 19) and student's t-test. One-way ANOVA and Tukey post-hoc tests were used to determine the statistical significance among groups. The data are presented as mean±SD (P <0.05).

BMSCs culture and cell markers expression
The isolated BMSCs after 3 passages Adipogenic differentiation was approved by oil red     O-positive cells, and osteogenic differentiation, by alizarin red S-positive cells (Figure 2).

In vitro PGC like cells differentiation from BMSCs
To achieve PGC like cells, BMSCs were treated with BMP4 for 4 days and characterized by morphological changes, Plzf immunostaining and real time PCR. After treatment, spindle -like BMSCs showed round shape and cluster accumulation ( Figure 3).
Immunocytochemistry test showed that 51.7% of BMP4 treated cells were positive for Plzf protein ( Figure 4).
Real time analysis showed that Nanog expression decreased significantly (P≤0.05) after

BMP4 treatment in PGC like cells in comparison
with BMSCs non-treated cells as control group.
Also, Dazl expression increased significantly (P

≤0.05) in PGC like cells after BMP4 treatment in
comparison with neonatal testis cells as control group ( Figure 5).

In vitro SSC like cells differentiation from
BMSCs-derived PGC like cells As described previously, BMP4 treatment was followed by RA cocktail for 14 days to induce SSC like cell differentiation. After this culture period RA treated cells showed morphological changes as shown in Figure 6.
Immunocytochemistry test showed that 54.06% of RA treated cells were positive for Stra8 protein as a specific marker after RA induction ( Figure 7).   (Figure 9).

In vitro derivation of SSC like cells has been
reported in multiple investigations (22,23   maintaining the proliferation of PGCs (26

Conflict of interests
The authors declare that there is no conflict of interest.

Funding
This work was supported by grants from Medical Faculty of Tarbiat Modares University, Tehran, Iran.